The Existence of a Soluble Plasmalogenase in Guinea Pig Tissues Christopher R. McMaster, Can-Qun Lu and Patrick C. Choy* Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada, R3E 0W3

The distribution of plasmalogenase for the hydrolysis of 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine

(plasmenylethanolamine) in the subcellular fractions of guinea pig tissues was examined. P l a s m a l o g e n a s e activity was found in high abundance in the cytosolic fractions of the brain and the heart. A s s e s s m e n t of microsomal

marker enzyme activities in the cytosolic fraction revealed that plasmalogenase activity in the cytosol was not due to microsomal contaminations. The characteristics of the cytosolie plnsmalogenase were very similar to the microsomal enzyme with respect to the pH profile of the reaction, the presence of divalent cations and Km values for plasmenylethanolamine. However, the cytosolic enzyme was slightly less stable at 55~ than the microsomal enzyme. Cytosolic enzyme activity was eluted as a broad peak in Sepharose 6B chromatography with an average molecular weight of 250,000. Our results demonstrate that most of brain plasmalogenase activity is soluble which makes the brain cytosol an excellent source to initiate the purification of this enzyme. Lipids 27, 945-949 (1992). Phospholipids containing an O-alkenyl group at the C-1 position (plasmalogens) are abundant in many mammalian tissues (1). The most widely distributed plasmalogens are the 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) species (1). Despite their ubiquitous distribution, only limited information is available on the metabolism or function of these phospholipids (2). The high concentrations of plasmenylethanolamines in electrically active tissues such as the brain and the heart imply that they may be involved in ion transport across membranes (3). Plasmalogens may also serve as reservoirs for prostaglandin precursors due to the large proportion of arachidonic acid at the C-2 position (4-6). Recently, a 1-alkenyl-2-acetyl-sn-glycero-3-phosphoethanolamine analogue of platelet activating factor has been identified in human neutrophils (7). The importance of plasmalogens to protect cell membranes from oxidative stress with the 1-alkenyl bond functioning as an oxygen radical scavenger has also been postulated (8). The 1-alkenyl bond of plasmenylethanolamine can be hydrolyzed by two separate catabolic pathways. In the mammalian brain and heart, a microsomal plasmalogenase has been identified that cleaves plasmenylethanolamine to lysophosphatidylethanolamine and a fatty aldehyde (9,10). An alternate route for the cleavage of the vinyl ether bond of plasmenylethanolamine involves the action of a putative phospholipase A2 followed by a lysoplasmalogenase. A lysoplasmalogenase activity has been characterized in both liver (11) and brain (12) microsomes. In view of the irreversible damage to both *To whomcorrespondenceshould be addressedat the Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0W3. Abbreviations: AU, absorbance unit; CDPethanolamine,cytidine diphosphoethanolamine; E1)TA, ethylenediaminetetraaceticacid; TLC, thin-layer chromatography.

cerebral and cardiac tissues by oxidative stresses during and after ischemia (13) and the protective role plasmalogens play in this process (8), the identification and characterization of plasmalogenase activities in these tissues is highly desirable. In this study, the distribution of plasmalogenase activity in the subcellular fractions of the guinea pig tissues was investigated. Surprisingly, plasmalogenase activity was found in high abundance in the cytosolic fraction from guinea pig brain and heart. The characteristics of the cytosolic enzyme activity were studied and compared to the activity found in the microsomal fraction.

MATERIALS AND METHODS Materials. Aldehyde dehydrogenase, NAD +, glutathione (reduced form), NADPH, cytochrome c (reduced form), iodine, potassium iodide, butylated hydroxytoluene, Triton QS-15, polyoxyethylene sorbitan monolaurate (Tween 20), and CDPethanolamine (cytidine diphosphoethanolamine) were purchased from Sigma Chemical Company {Toronto, Ontario, Canada). Thin-layer chromatography (TLC) plates (Sil-G25) were obtained from Fisher Scientific (Ottawa, Ontario, Canada). Silicic acid (BIO-SIL A) for column chromatography was a product of Bio-Rad Laboratories (Mississauga, Ontario, Canada). BDH Limited (Poole" England) provided the 2% dimethyldichlorosilanein 1,1,1trichloroethane solution. Phospholipid standards were purchased from Serdary Research Laboratories {London, Ontario, Canada). Sepharose 6B for column chromatography was a product of Pharmacia LKB Biotechnology (Uppsala, Sweden). CDP[1,2-14C]ethanolamine was a product of 1CN Biomedicals (Costa Mesa, CA). All other chemicals were of the highest grade available and were acquired from the Canlab division of Baxter Diagnostics Corporation (Mississauga, Ontario, Canada). Guinea pigs weighing 275 +_ 25 g were used throughout the study. Preparation of plasmalogens for enzyme assays. Plasmalogens were prepared as previously described (10). Briefly, lipids were extracted from porcine hearts by the method of Folch et al. (14) in the presence of 0.5% butylated hydroxytoluene (wt/vol). The volume was reduced in vacuo and the lipid sample was dissolved in chloroform and applied to a silicic acid column. The individual phospholipids were eluted from the column with increasing amounts of methanol in chloroform (15}. Fractions from the column were analyzed by TLC with a solvent containing CHC13/CH3OH/CH3COOH/H20 (70:30:2:4, by vol). The fractions containing diradylglycerophosphoethanolamine were pooled. Phoshatidylethanolamine in the pooled diradylglycerophosphoethanolaminefraction was destroyed by the preferential hydrolysis of the ester bonds of phosphatidylethanolamine by mild alkaline hydrolysis with 0.35 M NaOH in 96% methanol as described by Renkonen (16). Lysoplasmenylethanolaminewas prepared by hydrolyzing the diradylglycerophosphoethanolamine fraction in 0.35 M NaOH in 96% methanol for 45 min. The plasmenylethanolamine and lysoplasmenylethanolamine obtained after alkaline hydrolysis were repurified by LIPIDS, Vol. 27, no. 12 (1992)


C.R. McMASTERET AL. RESULTS AND DISCUSSION silicic acid column chromatography. The purity of t h e plasmalogen fractions was assessed by determining the ratio of the vinyl ether content and the total phosphorus Plasmalogenase assays. The disappearance of plasmenylcontent in the samples. Only preparations with purity ethanolamine was monitored by the loss of the vinyl ether bond. A typical assay resulted in the disappearance of greater than 96% were used for enzyme assays. Preparation of subcellular fractions from guinea pig 10-20 nmol of the vinyl ether bond of plasmenylethanoltissues. Guinea pig tissues were removed and placed on amine over the 15 rain incubation period. This change in ice The tissue was homogenized in 0.25 M sucrose/10 mM vinyl ether content resulted in an increase in absorbance Tris-HC1 (pH 7.4) with two 20 s bursts of a Polytron probe of 0.040-0.080 A.U. at 355 nm (Fig. 1) which could be (PT-30) (Brinkmann Instruments; Rexdale, Ontario, Can- easily detected by a modern spectrophotometer. Enzyme ada) at a speed setting of six. Alternatively, the brain tis- activity was linear with protein concentration between sues was homogenized in a Potter-Elvehjem homogenizer 0.5-3.0 mg of protein from guinea pig brain cytosol or equipped with a teflon pestle. The tissue homogenates microsomes. Subcellular localization of plasmalogenase activity. The were centrifuged at 10,000 X g for 20 min and the supernatant was centrifuged again at 100,000 • g for 60 rain. distribution of plasmalogenase activities in the subcellular The supernatant obtained from the last centrifugation fractions of guinea pig brain, heart and liver was inwas removed with a Pasteur pipette and designated as the vestigated. The enzyme activity was assessed by the discytosolic fraction. The precipitate containing the micro- appearance of the substrate during the reaction. A small somal pellet was dispersed in the homogenizing buffer with amount of plasmalogenase activity was found in the mitochondrial fraction of the brain and heart (data not a Dounce homogenizer equipped with a type A pestle Plasmalogenase assay. All glassware including test shown); however, most of the enzyme activity (>90%) was tubes were treated with a 2% dimethyldichlorosilane in located in the microsomal and cytosolic fractions. As 1,1,1-trichloroethane solution to minimize the adherence depicted in Table 1, 83% of the total brain enzyme activof lipids to glass containers. Plasmalogenase activity was ity was found in the cytosolic fraction, whereas 68% of monitored by the disappearance of the substrate (9,10). t h e total heart enzyme was located in the cytosol. A Purified plasmenylethanolamine (2 ~mol) was suspended similar distribution of plasmalogenase activity was also in 1 mL of 10 mM Tris-HC1 (pH 7.4) containing 0.05% observed in the subcellular fractions of the rat brain and TWeen 20. The mixture was sonicated in a water bath until heart (data not shown). The distribution of enzyme activtranslucent. The reaction mixture (1.5 mL) contained ity between the cytosol and microsomes was not affected 300 nmol dispersed plasmalogen, 50 mM Tris-HC1 (pH 7.4) by the mode of tissue homogenization. Plasmalogenase and an enzyme preparation containing 0.75-1.0 mg of pro- activity in the liver had a similar subcellular distribution, tein. The reaction was initiated by the addition of the en- but the activity was very low. In order to determine if the hydrolysis of the vinyl ether zyme, and the mixture was incubated at 37~ for 15 rain. Control tubes contained either no enzyme or enzyme that group of the plasmenylethanolamine by guinea pig brain had been incubated at 100~ for 5 rain. At 0 and 15 min cytosol was due to the direct acton of the plasmalogenase of incubation, 600 gL was removed from the reaction mix- or the combined action of phospholipase A2/lysoplasture and placed into a tube containing 1.5 mL CHC13/ malogenase, the reaction mixture was analyzed for the CHaOH (2:1, vol/vol). Water and chloroform were added disappearance of plasmenylethanolamine as well as the to cause phase separation. The upper phase was removed, formation of the radylglycerophosphoethanolamine. The and an aliquot of the lower phase was assayed for vinyl reaction mixture contained 300 nmol of plasmenylethanolether content spectrophotometricaUy at 355 nm. Plas- amine, and the assay for plasmalogenase activity was carmalogenase activity was calculated from the difference ried out as described in the preceding section. The reacin vinyl ether content between the 0 and 15 rain time tion was terminated by the addition of 3 mL of CHC13/ points. Total enzyme activity was calculated from the CH3OH (2:1, vol/vol), and the lipids in the lower phase product of the specific activity of the enzyme and the total protein content in the subcellular fraction. The determination of marker enzyme activities. The degree of microsomal contamination in the cytosolic frac0.8q tion was determined by the activities of known micro~O ~O ~Q somal marker enzymes in the cytosol. NADPH-cyto0.6 chrome c reductase (17), phosphoethanolaminetransferase activities (18) and 5'-nucleotidase activities (19) were used 0.4 as microsomal markers. Analytical procedures. Lipid phosphorus was assayed ~176 0.2 by the method of Bartlett (20). Plasmalogen (vinyl ether) content in the sample was assessed by the method of Gottfried and Rapport (21) based on iodine determination. The 0.0 25 50 75 100 125 correction factor for alkylacyl compounds was not used pl~malc,g~n (r,mo0 for the calculation of vinyl ether content. Protein was determined by the method of Lowry et at. (22). All results FIG. 1. The relationship of plasmenylethanolamine content v s . abare expressed as the mean ___standard deviation of at least sorbance by the determination of the vinyl ether group. Each point t h r e e separate experiments except where otherwise in- is the mean of four separate experiments. An identical curve was dicated. The points on all figures have standard deviations obtained when lysoplasmenylethanolamine was used as the vinyl ether source. of less than 15% of the mean. LIPIDS, Vol. 27, no. 12 (1992)




o ,~ 40

PlasmalogenaseActivities in the SubcellularFractions of G u i n e a P i g T i s s u e s a

Tissue Brain Cytosolc Microsomes c Cytosold Microsomes d Heart Cytosol Microsomes Liver Cytosol Microsomes

Specific activity Plasmenylethanolamine disappearance (nmol/h/mg protein) 89.8 66.1 84.2 77.8

-+ 7.3 +_ 3.6 +_ 7.9 + 13.1

Total activity b Distribution (%) 83% 17% 78% 22%

47.6 + 5.8 57.0 +-- 8.1

68% 32%

6.5 _+ 2.5 5.1 _ 1.5

75% 25%

a Plasmalogenase activities were determined by substrate disappearance as described in Materials and Methods. Each value represents the mean +_ standard deviation of at least four different experiments. bTotal activity was calculated from the specific activity of the enzyme and the amount of protein in each subcellular fractions. c Prepared by homogenization with a Polytron generator equipped with a PT-30 probe. dprepared by homogenization with a Potter-Elvehjem homogenizer equipped with a teflon pestle.

"8 o




~= 20 o /



~ 15

O 30 T~e(~


O 60

FIG. 2. Time course for the hydrolysis of plasmenylethanolamine and lysoplasmenylethanolamine in guinea pig brain cytosol. Enzyme activities {1.25 mg cytosolic protein) were assayed at 37~ in the presence of 300 nmol plasmenylethanolamine ( 9 ) or lysoplasmenylethanolamine (O) by measuring suhstrate disappearance as described in Materials and Methods. Each point represents the mean of three separate experiments.

TABLE 2 The Distribution of Microsomal Marker Enzyme Activities in the Microsomal and Cytosolic Fractions of Guinea Pig Brain and Heart

Percentage distribution a Brain Heart

Enzyme NADPH-cytochrome c reductase

were separated by TLC with a solvent containing CHC13/ CH3OH/CH3COOH/H20 (70:30:2:4, b y vol). At the 0 min time point (control), no radylglycemphosphoethanolamine fraction was detected after TLC separation. After 15 min of incubation, 22 + 3 nmol of plasmenylethanolamine was hydrolyzed with the formation of 14 +_ 2 nmol of radylglycerophosphoethanolamine. Analysis of the vinyl ether content in the radylglycerophosphoethanolamine fracton revealed t h a t there was no detectable vinyl ether group in this fraction. Our results indicate t h a t the m a j o r i t y of the plasmenylethanolamine was hydrolyzed by plasmalogenase and not by a phospholipase A2/lysoplasmalogenase system. To further confirm the above results, t i m e courses for the hydrolysis of plasmenylethanolamine and lysoplasmenylethanolamine in guinea pig brain cytosol were dete~ mined (Fig. 2). The hydrolysis of plasmenylethanolamine was linear up to 30 min. However, the brain cytosol had very limited ability to hydrolyze lysoplasmenylethanola m i n e The results obtained in this s t u d y s u p p o r t the notion t h a t the hydrolysis of plasmenylethanolamine resulted from the direct action of plasmalogenase in the brain cytosol. I t could be argued t h a t the plasmalogenase activity in the brain and heart cytosol m i g h t arise from the cont a m i n a t i o n of microsomal particles. Hence, the microsomal m a r k e r enzyme activities in the cytosolic fractions were assessed (Table 2). The result shows t h a t the contamination of the cytosolic fraction by microsomal enzyme m a r k e r s did not exceed 12% in any of the cases. Hence, the plasmalogenase activities in the cytosolic fraction of the brain and heart could not arise solely from microsomal contamination.


Cytosol Microsomes 5'-Nucleotidase Cytosol Microsomes Phosphoethanolamine transferase Cytosol Microsomes

12 88 --6 94

--2 98 N.D. b 100

aEach value is the average of three separate experiments. bN.D., not detectable.

Characterization of plasmalogenase activities. In view of the abundance of soluble plasmalogenase in the brain, i t s cytosol was employed for further characterization of the enzyme activity. The effect of substrate concentrations on plasmalogenase activity was investigated (Fig. 3). F r o m the double reciprocal plot of enzyme activity vs. plasmenylethanolamine concentrations, the K m of the enz y m e for plasmenylethanolamine was e s t i m a t e d to be 154 gM. The Km of p l a s m e n y l e t h a n o l a m i n e for the cytosolic enzyme is comparable to the microsomal enzyme (105 ~M) and also the enzyme obtained from an acetone extract of the bovine brain (285 gM) (9). Similar to the microsomal enzyme (data not shown), the cytosolic enzyme from the brain was also inhibited by 1 m M ethylened i a m i n e t e t r a a c e t i c acid (EDTA) a n d 1 m M M n 2+. However, the presence of 1 m M Ca 2+ or M g 2+ had v e r y little effect on either enzyme activity (Table 3) (10). Taken together, our results show t h a t plasmalogenase activity in the cytosol m a y require a m i n i m u m level of metalic cation(s) for full activity. The p H profiles of the brain cytosolic and microsomal plasmalogenase activities were determined (Fig. 4). B o t h enzymes displayed similar p H profiles with o p t i m a at p H 7.5. W h e n the enzymes from LIPIDS, Vol. 27, no. 12 (1992)




@ ~

@------~ @

t~o 60


t h e m i c r o s o m a l a n d c y t o s o l i c f r a c t i o n s were i n c u b a t e d a t 5 0 ~ a n d 5 5 ~ {Fig. 5), t h e m i c r o s o m a l e n z y m e w a s m o r e s t a b l e t h a n t h e c y t o s o l i c form.

Gel filtration chromatography of guinea pig brain cytosol. G el f i l t r a t i o n c h r o m a t o g r a p h y w as e m p l o y e d to

~ 40

9 0.04 ~0.00 -0.02 0.00





1/[pl~ffienyictt~ola~ael (~M"1)

/ i

05 0




1O0 150 200 [Pl~me~ylethano]amine I (#M)



FIG. 3. Effect of substrate concentrations on guinea pig brain cytosolic plasmalogenase activity. Plasmenylethanolamine (2 banol) was suspended in 1 mL of 10 mM "I~i~HCI]0.05% Tween 20 by sonication and appropriate aliquots of the suspension were added to the enzyme assays. Each assay contained 1.25 mg of cytasolic protein and the reaction was carried out at 37~ for 15 min as described in Materials and Methods. Inset is the Lineweaver-Burke plot of the same data. Each point represents the mean of three separate determinations.

c o n f i r m t h e t r u e solubility of t h e brain cytosolic plasmalogenase. T h e b r a i n c y t o s o l (2 mL) w as a p p l i e d t o a S e p h a rose 6B c o l u m n (3 • 48 cm) e q u i l i b r a t e d w i t h 0.1 M TrisHC1 (pH 7.5}. S u b s e q u e n t to s a m p l e a p p l i c a t i o n , t h e colu m n w a s w a s h e d w i t h t h e s a m e buffer, a n d f r a c t i o n s of 1.2 m L were c o l l e c t e d a n d a s s a y e d for e n z y m e a c t i v i t y {Fig. 6). P l a s m a l o g e n a s e a c t i v i t y w a s e l u t e d as a b r o a d p e a k away f r o m t h e v o i d v o l u m e of t h e c o l u m n . T h e fraction with the highest enzyme activity had an apparent m o l e c u l a r w e i g h t of 250,000. T h e b r o a d a n d a s y m m e t r i c a l


g ~ 75 E


~ 5O :E




t ~O0

v x-

.:'.,. :'.:"







0 w









~ iio: ,




:'. ~,~

0.1 0.0


Fraction N u m b e r







FIG. 4. The pH profile of guinea pig brain cytasolic and microsomal plasmalogenase activities. Enzyme activities in the cytosolic (O) and microsomal (A) fractions were assayed by substrate disappearance as described in Materials and Methods section. Each assay contained 1.25 mg cytasolic protein or 0.8 mg micrasomal protein and the reactions were incubated at 37~ for 15 min. Tris~succinate buffer was used from pH 6-7, and Trls-HCI was used from pH 7-10. No significant difference was detected in enzyme activity at pH 7 when either buffer was utilized. Each point represents the mean of four separate experiments. LIPIDS, Vol. 27, no. 12 (1992)

FIG. 6. Sepharose 6]] chromatography of plasmalogenase activity in guinea pig brain cytasoL Guinea pig brain cytasol (2 mL) containing 16 mg of protein was applied to a Sepharose 6B column (3.0 X 48 cm) equilibrated with 100 mM Tris-HCl (pH 7.5). Subsequent to sample application, the coblmn was washed with the same buffer and 1.2 mL fractions were collected. An aliquot (0.8 mL) of the fraction was used for enzyme activity determination and the activity is expressed as nmol/h/mL. The total yield of plasmalogenase activity after column chromatography was 41 4" 5% of the enzyme activity applied to the column. The void volume of the column is indicated by V o. Thyroglobulin (669 kDa), ferritin (440 kDa) and aldolase (158 kDa) were used for column calibration.


CYTOSOLIC PLASMALOGENASE p e a k of enzyme activity implies t h a t the e n z y m e was eluted as multimeric proteins or in aggregation with other cytosolic proteins. An alternate explanation is t h a t the enzyme m i g h t still be associated with some lipid molecules present in the cytosol. The fact t h a t none of the enzyme activity was eluted near the void volume of the column suggests t h a t it was not complexed with microsomal or large liposomal particles. In a previous study, the existence of a metabolite in the rat brain cytosol for the elimination of the vinyl ether bond of plasmenylethanolamine was reported (23). The material was t h e r m o s t a b l e at 100~ had a low molecular weight and was found to be a non-protein e n t i t y which was later identified as ascorbic acid (24). Hence. our s t u d y is the first identification of a truly soluble plasmalogenase from mammalian sources. In the last two decades, the existence of plasmalogenase activity in the brain has been a matter of debate. Plasmalogenase activity was identified in the microsomes of rat brain (9), in neuronal perikarya, astroglia, and oligodendroglia from bovine brain (25), and from the brains of rats and monkeys (26). Using another approach, it was shown t h a t the rat brain had no ability to catabolize radiolabeled plasmalogens, b u t low levels of lysoplasmalogenase activity were detected in brain (12}. Our results support the existence of considerable amounts of plasmalogenase activity in the brain and also the presence of low levels of lysoplasmalogenase activity. A t present, the reason for the discrepancy in the identification of plasmalogenase activity is not entirely clear. One explanation is t h a t the activity of the plasmalogenase is highly dependent on the source of the plasmalogen and the m e t h o d of suspension in the buffer. The assay procedure in this s t u d y had been optimized to provide a high plasmalogenase activity. The similarity in characteristics between the cytosolic and microsomal enzyme m a k e s it plausible to speculate t h a t b o t h enzymes m a y originate from the s a m e protein. However, the ability to obtain the s a m e distribution of enzyme activity between the two c o m p a r t m e n t s b y different m e t h o d s of homogenization confirms t h a t the cytosolic enzyme was not mechanically detached from the microsomes during tissue homogenization. The identification of a t r u l y soluble plasmalogenase makes the guinea pig brain cytosol an ideal source of the enzyme for its subsequent purification. At present, the physiological significance of the distribution of the e n z y m e in two subcellular c o m p a r t m e n t s remains undefined.

ACKNOWLEDGMENTS This study was supported by the Medical Research Council of Canada. CRM is a recipient of an MRC Studentship Award. REFERENCES 1. Horrocks, L.A., and Sharma, M. (1982) in Phospholipids (Hawthorne, J.N., and Ansell, G.B., eds.) pp. 51-93, Elsevier Biomedical Press, Amsterdam. 2. Paltanf, F. (1984) in Ether Lipids (Mangold, H.K., and Paltauf, F., eds.) pp. 211-230, Academic Press, New York. 3. Gross, R.W. (1984) Biochemistry 23, 158-165. 4. Gross, R.W. (1985) Biochemistry 24, 1662-1668. 5. Wykle, R.L., Blanl~ M.L., and Snyder, E (1973)Biochim. Biophys. Acta 326, 26-33. 6. Horrocks, L.A., and Fu, S.U. (1978) Adv. Exp. MedL BioL 101, 397-406. 7. Tessner, T.G., and Wykle, R.L. (1987) J. Biol. Chem. 262, 12660-12664. 8. Morand, O.H., Zoeller, R.A., and Raetz, C.R.H. (1988) J. Biol. Chem. 263, 11597-11606. 9. D'Amat~ R.A., Horrocks, L.A., and Richardson, K.E. (1975) J. Neurochem. 24, 1251-1255. 10. Arthur, G., Covic,L., Wientzek, M., and Choy, PC. (1985)Biochim. Biophys. Acta 833, 189-195. 11. Alexander-Jurkowitz, M., Ebata, H., Mills, J.S., Murphy, E.J., and Horrocks, L.A. (1989)Biochirr~ Biophys. Acta 1002, 203-212. 12. Gunawan, J., and Debuch, H. (1982)J. Neurochem. 39, 693-699. 13. Katz, A.M., and Messineo, F.C. (1981) Circ. Res. 4R 1-16. 14. Folch, J., Lees, M., and Sloane~Stanley, G.H. (1957)J. Biol. Chem. 226, 497-506. 15. Sweeley, C.C. (1969) Methods Enzymol. 14, 254-267. 16. Renkonen, O. (1963) Acta Chem. Scand. 17, 634-640. 17. Masters, B.S~, Williams, Jr., C.H., and KamJn, H. (1967)Methods EnzymoL 10, 565-569. 18. O, K-M., Siow, Y.L., and Choy, PC. (1989) Biochem. Cell Biol. 67, 680-686. 19. Bers, D.M. (1979) Biochim. Biophys. Acta 555, 131-146. 20. Bartlett, G.R. (1959) J. Biol. Chem. 234, 466-468. 21. Gottfried, E.L., and Rapport, M.M. (1962) J. Biol. Chem. 237, 329-333. 22. Lowry, O.H., Roseborough, M.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 23. Yavin, E., and Gatt, S. (1972) Eur. J. Biochem. 25, 431-436. 24. Yavin, E., and Gatt, S. (1972) Eur. J. Biochem. 25, 437-446. 25. Dorman, R.V., Toews, A.D., and Horrocks, L.A. (1977) J. Lipid Res. 18, 115-117. 26. Ansell, G.B., and Spanner, S. (1968) Biochem. J. 108, 207-209. [Received June 4, 1992, and in revised form August 20, 1992; Revision accepted September 24, 1992]

LIPIDS, Vol. 27, no. 12 (1992)

The existence of a soluble plasmalogenase in guinea pig tissues.

The distribution of plasmalogenase for the hydrolysis of 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) in the subcellular ...
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